Antibody-based therapeutics have played an important role in targeted therapy for various disorders, such as cancers and immunological diseases. In recent years, antibody drug conjugates (ADC) have been explored extensively for effective delivery of drugs to target sites. For example, chemical modification has been widely used for conjugating drugs to antibodies either through lysine side chain amines or through cysteine sulfhydryl groups. However, these conjugation methods frequently led to a heterogeneous mixture of conjugates having different molar ratios of drug to antibody, non-specific conjugation sites, as well as different efficiency, safety, and pharmacokinetics. See Tanaka et al, FEBS Letters 579:2092-2096 (2005). Reactive cysteine residues engineered at specific sites of antibodies for specific drug conjugation with defined stoichiometry has also been made. See Junutula et al., Nature Biotechnology, 26: 925-932 (2008). However, expression and conjugation of such cysteine engineered antibodies and antibody-drug conjugates require lengthy and complicated reaction procedures. See, e.g., Gomez et al., Biotechnology and Bioengineering, 105(4): 748-760 (2009). Antibody aggregates may also be generated during the process of making the cysteine engineered antibodies and the antibody-drug conjugates. Unnatural amino acid residues have also been incorporated into antibodies as chemical handles for site-specific conjugation. See Axupa et al., PNAS, 109: 16101-16106 (2012). To implement this methodology, an orthogonal pair of amber suppressor tRNA and aminoacyl-tRNA synthetase has to be integrated into an expression host first. Then, the mutant antibody can be expressed in this special host with medium supplement of the unnatural amino acid. This process is not only time consuming, but also very low in antibody expression yield.
Recently, enzymatic approaches to making ADCs using a transglutaminase have been explored. Transglutaminases (TGase) transfers a moiety having an amine donor group to an acceptor glutamine residue through transglutamination. Full-length IgG antibodies of human isotype contain a conserved glutamine residue at position 295 of the heavy chain (Q295). Because this glutamine residue is in close proximity to an N-glycosylation site (N297), it was generally believed that Q295 on the full length antibody is inaccessible to TGase when the antibody is N-glycosylated. To allow TGase acting on full length antibodies, the Fc region of the antibody was deglycosylated or mutated to remove the N-glycosylation site prior to the TGase-mediated conjugation. See WO2013/092998. Alternatively, glutamine-containing sequence “tags” have been inserted into the antibodies' light or heavy chains to provide acceptor glutamine sites. See WO2012059882. Hence, all current site-specific ADC technologies rely on engineered antibody mutants, which may result in potential immunogenicity and in vivo instability. There is a strong need for an efficient site-specific antibody conjugation tool where intact antibody can be used directly.
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